Nonalcoholic fatty liver disease (NAFLD) comprises a spectrum of liver pathologies from simple steatosis to nonalcoholic steatohepatitis (NASH; hepatic inflammation and fibrosis) and cirrhosis. NAFLD affects greater than 30% of the general US adult population, and disturbingly, prevalence rates of NAFLD and NASH appear to be greater in the military and veteran population. NASH is directly linked to increased liver-related, cardiovascular disease and all-cause mortality and also the need for liver transplantation. Previous studies from our group and others indicate that mitochondrial dysfunction is linked to NASH development and progression. Unfortunately, mechanism(s) that regulate hepatic mitochondrial function and increased susceptibility to NASH are largely unknown. Our group has recently demonstrated that normal endothelial nitric oxide synthase (eNOS) activation is lost in an obese rat model during the transition to NASH, and that systemic NOS inhibition causes hepatic mitochondrial dysfunction and accelerates NAFLD progression to NASH. These studies form the overall hypothesis of this proposal, that hepatic eNOS and eNOS-derived nitric oxide (NO) are critical in maintaining normal hepatic mitochondrial function and quality control in the prevention of NASH. While it is well established that eNOS and NO regulate peroxisome proliferator-activated gamma co-activator alpha (PGC-1?), a co-activator of nuclear transcriptional factors that control mitochondrial biogenesis, we have recently collected novel preliminary data that eNOS deficiency also causes a dramatic reduction nuclear factor-E2-related factor-2 (NRF2/NFE2L2) and in markers of hepatic autophagy and mitophagy, the cellular processes responsible for clearance of damage organelles and mitochondria. The loss of mitophagy in eNOS deficiency occur in conjunction with increased H2O2 emission, reduced anti-oxidative capacity, and susceptibility to western diet (high fat, sucrose, cholesterol) induced NASH. We will utilize dietary, pharmacological, and in vivo and in vitro molecular approaches (gain and loss of function studies) to mechanistically examine the novel links between hepatocellular eNOS and mitophagy in the development and progression of NASH. The specific aims will: (1) determine the role of hepatocellular eNOS in susceptibility to NASH, (2) test if targeting NRF2 and BNIP3 increases mitophagy and rescues NASH, and (3) test if upregulation in mitochondrial-targeted antioxidant defense alleviates western diet induced NASH. These studies will provide insight into reducing the incidence of NASH in our Veteran population.